Successful biotechnology researchers: The Nano3Bio consortium during its public conference ‘The Future of Chitosans’ in Hyderabad. [Click image to load full size.]

Since fossil raw materials run out with a more or less dramatic need to be substituted, renewable resources are becoming increasingly important. In future, the biological production of raw materials has to play an even greater role. The international project Nano3Bio now contributed to fulfil this challenge. Nano3Bio’s main goal was the biotechnological production of so-called chitosans, which are used as raw materials for medicine, agriculture, cosmetics and other application fields. The European Commission supported the research project with almost 9 million Euros over four years. Recently the consortium held its final conference ‘The Future of Chitosans’ in Hyderabad, India. “We’re proud of Nano3Bio’s outcomes. In important fields the consortium achieved or prepared a breakthrough from basic research to biotechnological applications”, says Professor Dr. Bruno Moerschbacher, biologist at the University of Münster and coordinator of the project.

For example within the Nano3Bio project researchers discovered a protocol for production of chitosans with defined structures, they developed a low-cost protein engineering technology and they isolated and identified the first chitosans generated by microalgae. Moreover the project brought out significant research results on the internalisation of chitosan nanocapsules into human cells, which is especially relevant for therapies of cancer with chemotherapeutics and could lead to more effective therapies with reduced adverse effects and better life quality for patients. Nano3Bio identified genes from different organisms (bacteria, fungi, algae) that can be used to drive the biotechnological production of encoded enzymes. These were then characterised and used for the biotechnological conversion of chitin into novel high quality chitosans. Furthermore, the project developed so-called electrospun chitosan nanofibers and electrosprayed chitosan nanoparticles as technological platforms for the encapsulation and efficient release of bioactives, vaccines and drugs in human medicine. And it invented thermo-sensitive chitosan hydrogels which are promising materials for regenerating damaged tissues. Moerschbacher: “Some of these achievements include huge economic potential.”

Not least, the project underlined its emphasis on sustainability by performing the first detailed life cycle assessment of chitosan production in order to evaluate and to compare newly invented approaches to traditional ones in terms of their environmental impact, e.g. concerning topics such as greenhouse-gas emissions, water use or land use. And Nano3Bio expressed policy recommendations based on a regulatory analysis for the field of nano-biotechnology and chitosans.

“These are impressive results. However, the future road still appears to be challenging. For example, it will be important to further determine which biological organisms are able to produce exactly that quality and quantity of chitosan required for specific applications”, Moerschbacher states. The researchers assume that many other fields of application will be found in which a specific chitosan can replace or support other substances. This is desirable, since one of the good qualities of chitosans lies in the fact that they are tolerated by the human body and biodegradable in the environment.

Selected Nano3Bio major achievements

New method for chitosan quantification developed

The University of Münster developed a chitosan quantification method that allows measuring the amount of chitosan within unknown samples in the microgram range. To this end the chitosan polymers in the sample are in a first step converted to smaller oligomers using a cocktail of different recombinant and well-characterised enzymes. In a second step these oligomers are analysed by mass spectrometric measurements in combination with light scattering detection. With the help of internal standards this allows estimating the original amount of chitosan in the sample. The availability of this method will highly improve the quality control of novel chitosans produced within the Nano3Bio project and it allows monitoring the efficiency of chitosan production processes. Moreover it will support the characterisation of chitosan modifying enzymes and might also help to optimize the production of chitosan-based nanomaterials developed by the Nano3Bio consortium.

(Nano3Bio consortium partner in charge: University of Münster, Germany)

Regenerative medicine is a growing area, constantly in search for new materials where cells proliferate and regenerate damaged tissues. In this regard, thermo-sensitive chitosan hydro-gels with cell encapsulation properties are promising materials for regenerating tissues and they are less invasive than currently applied techniques. Mimicking the 3D environment of cells in vivo is a key factor that should complement the appropriate tissue-like properties. One of the most promising materials to resemble these natural environments are hydrogels. Hydrogels are polymeric networks with the ability of imbibing large amounts of water or biological fluids. In the frame of the Nano3Bio project, chitosan hydrogels for cell encapsulation have been developed. These hydrogels are designed to undergo a rapid phase transition from liquid to solid in response to temperature changes. The thermo-sensitivity of these hydrogels makes them ideal as injectable materials in the biomedical field due to their gelation at human body temperature (37ºC). Moreover, when cells are incorporated in the chitosan solution they get encapsulated in the hydrogel during gelation. The ability of these hydrogels to imbibe the cells maximises the cyto-compatibility and minimises the number of hydrogel processing steps.

Chitosan binds a red dye in unique and intriguing ways

Researchers from the University of Münster investigated how long chain-chitosan molecules interact with much smaller ones of a red dye called ‘cibacron brilliant red’ (CBR) when they are mixed in water. The goal behind this was to further investigate the nature of a sensitive analytical method to determine the concentration of chitosan in different types of products and environments (e.g. cosmetics, pharmaceutical formulations, foods). To this end, the researchers set up a panel of various instrumental techniques using different forms of light (visible, fluorescent and so-called dichroic). When the chitosan chains associate with those of the dye, they form very small particles whose sizes can be tuned-up in the range of 200 to 2000 nm, depending on the ratio of chitosan to CBR. Since it is difficult to observe the particles that are formed directly, the techniques mentioned above study the interaction indirectly and they allow the researchers to generate assumptions regarding their structure. The results of these studies suggest that at molecular level helices of chitosan in which the amino groups are oriented at opposite sides of the chain axis as well as stacked dye molecules. Previous computer modelling simulations performed in other studies predicted this possibility and now the University of Münster provided compelling experimental evidence. This rather particular form of interaction makes CBR ideal for the determination of chitosan concentration. Future studies will explore if the interaction and the techniques used within the survey are sensitive to investigate factors besides the concentration of chitosan. One particular factor that could be studied is the pattern of acetylation of chitosan (whether ‘random’ or ‘blockwise’), which is fairly difficult to study at the moment and might be analysed more efficiently using this technique.

(Nano3Bio consortium partner in charge: University of Münster, Germany)

Self-assembled xanthan-COS nanofibers

Molecular self-assembly is the process of spontaneous organization of molecules into ordered structures by non-covalent interactions. This self-assembly process has been proved to be efficient for developing bio-inspired three-dimensional nanostructures with enhanced complexity and functionality. For instance self-assembled nanostructures could be used as carriers for drug delivery, as hydrogels for cell culture and tissue repair, amongst others. Within the scope of the Nano3Bio project the Technical University of Denmark (DTU) demonstrated for the first time that xanthan gum and low molecular weight chitosans can be used as building blocks to generate self-assembled nanofibers by polyelectrolyte complexation in dilute regimes. Nano-fibres with fine-tuned properties made of these two unique polymers were created using mild conditions by exploring different mixing conditions. These systems were proved to be efficient in encapsulation and delivery of pharmaceuticals.

(Nano3Bio consortium partner in charge: Technical University of Denmark, DTU)

Policy recommendations generated based on regulatory analysis

The Nano3Bio project aims at a breakthrough from basic research to biotechnological production of chitosans. Within this EU-funded project Perseus compared regulatory aspects for conventionally produced chitosans with biotechnology based ones, addressing the following three areas:

Regulatory requirements for operations,

Regulatory requirements for products,

Regulatory gap/bottleneck analysis.

The results of a regulatory analysis undertaken by Nano3Bio were published in the paper "Biotechnologically produced chitosan for nanoscale products. A legal analysis" (2018). Read paper or download PDF ...

On the operational side, no particular bottlenecks regarding the national implementations of Directives covering operational aspects were found.Regarding product legislation, no new EU legislation for nanomaterials has been introduced. Either provisions are included in other legislation or introduced through national legislation. The main issue raised in this context is the difficulty of applying the nanomaterial definition to complex and diverse products such as chitosan, where some forms may be considered nanomaterials and others pure chemicals. Analysing the requirements for specific market introductions (including products for medical, cosmetic or agricultural use) reveals challenges:

Many of the product legislations incorporate provisions addressing nanomaterials.

When this is the case, this usually results in additional data requirements and/or a default higher risk classification.

The indication ‘nano’ on labels is required in the area of food, cosmetics and biocides.

In order to stimulate the use of biotechnology, the following recommendations were formulated:

Electrospun and electrosprayed chitosan nanofibers and particles developed

Within the scope of the EU funded Nano3Bio project the Technical University of Denmark (DTU) has been working on the development of functional nano-formulations of chitosans. In particular DTU developed electrospun chitosan nanofibers and electrosprayed chitosan nanoparticles as technological platforms for the encapsulation and efficient release of bioactives, vaccines and drugs. The accordant main achievements include:

Chitosan-phospholipid electrospun hybrid nanofibers stable at aqueous systems that can be used as a drug delivery matrix for biopharmaceutical applications, as well as scaffolds for tissue engineering applications.

Electrospinning and electrospray technologies are straight forward, cost-effective and scalable techniques suitable for the development of continuous and highly functional nanostructures, such as nanofibers, nanobeads, nanorods, nanotubes and nanospirals from a wide range of polysaccharides, proteins or lipids. Nanostructures with added functionalities can be achieved e.g. through utilization of blends, coaxial core-shell spinning, inclusion of other functional molecules and particles, or through the adsorption of functional components to surfaces. In the areas of bio-pharmaceutical applications, key advantages of electrospun and electrosprayed nanostructures are their large surface-to-volume ratio (e.g. allowing extensive interactions with the surrounding environment), their high encapsulation efficiency as well as their versatility to encapsulate drugs with different properties.

(Nano3Bio consortium partner in charge: Technical University of Denmark, DTU)

Biotechnology provides novel chitosans

Chitosans are a promising class of biopolymers with many potential applications, but the chitosans commercially available today often do not fulfil the requirements for sensitive markets such as pharmaceutics or cosmetics. One problem lies in batch-to-batch differences typically observed with chitosans produced from shrimp shell chitin, a waste by-product of shrimp peeling factories. Also, the animal origin of these chitosans is sometimes considered as problematic. Therefore, the Nano3Bio consortium aims to produce well defined chitosans with known structures and functionalities through biotechnological approaches. The tools required for this approach come from nature itself, namely enzymes such as chitin synthase which produce chitin from small sugar molecules, and chitin deacetylases which convert chitin into chitosans. Work at the University of Münster, the co-ordinating partner of the Nano3Bio project, has now identified a number of genes from different organisms - bacteria, fungi, viruses, algae - that appear to code for chitin deacetylases. These genes were used to drive the biotechnological production of the enzymes they encode. The recombinant enzymes were then characterised and used for the biotechnological conversion of chitin into chitosan. Interestingly, the chitosans obtained differ in their fine structure from all currently available chitosans which invariably are produced from chitin using chemical methods. Clearly, the chemical process yields “simple” chitosans which differ from the more complex and more varied chitosans found in nature. Ongoing work in the Nano3Bio project aims to test the material properties as well as the biological activities of these novel, “third generation” chitosans.(Nano3Bio consortium partner in charge: University of Münster)

Learn more about this Nano3Bio achievement from project coordinator Bruno Moerschbacher in the short video below:

First detailed life cycle assessment of chitosan production

The Nano3bio project has carried out the first detailed life cycle assessment (LCA) of chitosan production from crustacean shells, using data from two manufacturers in India and Germany. LCA assess the overall environmental impacts caused by a system of production, use, and disposal processes necessary to provide a specific product. The LCA study within Nano3Bio includes the production and processing of all involved raw materials (crustacean shells as a by-product from fisheries), production of materials and energy carriers (chemicals, fuels, electricity) used in the manufacturing process, and the disposal of waste generated in the process (solid waste and wastewater). Knowledge gained from this LCA will be used by the manufacturers to define strategies to reduce their environmental impacts, both in their directly controlled activities as well as in their supply chain. The results from this study will be submitted to the European LCA database ELCD, where they will be publicly available. Implementing LCA during research activities is an important approach within the Nano3Bio project in order to contribute to an overall sustainable development regarding the usage of raw organic materials.(Nano3Bio consortium partner in charge: 2.0 LCA Consultant APS)

Internalisation of chitosan nanocapsules into human cells

Drug administration to patients is frequently associated with adverse effects. This is especially relevant for therapies of cancer with chemotherapeutics leading to reduced life quality of the patient. Adverse effects are mostly related to the inefficient deliverance of drugs to the tumour and thus to systemic dissemination affecting the whole body. In the past, various strategies have been developed to improve tumour targeting and thus to minimize adverse effect. One of the most promising approaches is the encapsulation of therapeutics into particularly small sized carriers. The development of such carriers has been intensively followed in the last decades. Today, novel chitosan-based nanocapsules represent an achievement of the Nano3Bio consortium. The chitosan applied in this context is a fully degradable biopolymer preventing the accumulation of the capsules in the human body. The efficient uptake of the chitosan-nanocapsules into certain cells due to their unique physicochemical properties envisions an improved targeting of tumours and thus a reduction of adverse effects during cancer therapies. The University of Heidelberg (Germany) mainly carried out the research and development activities related to chitosan nanocapsules.(Nano3Bio consortium partner in charge: University of Heidelberg)

Learn more about this promising Nano3Bio achievement from the following short video:

First microalgal chitosan isolated and identified

The research and development team of Greenaltech, a Nano3Bio partner company from Spain, has recently discovered the presence of natural chitosans in certain green microalgae species. With the help of other Nano3Bio consortium partners, Greenaltech is currently working on their characterization. The microalgal chitosans are completely natural sub-stances that do not suffer from any chemical modifications. Moreover, they are of non-animal origin, an important advantage in some industries from a regulatory and marketing perspective. Furthermore, the microalgal chitosan production process is fully controllable as it is performed in closed reactors from the inoculation of the culture media with microalgae to the last chitosan purification step. These advantages, together with the physico-chemical and bioactive properties that are still being elucidated within the Nano3Bio consortium, will be taken into account to select the niche markets in which the microalgal chitosans may have a higher potential of success.(Nano3Bio consortium partner in charge: Greenaltech)

Get a brief introduction about this process from the following short video:

Chitosan-based hand cream formulation developed

For the cosmetic sector, chitosans are especially interesting due to their antimicrobial and thickening properties. Besides other functions, they can be used as a multifunctional raw material covering the two requirements of microbial stability and viscosity control with just one ingredient. These two functions are determined by variable chitosan characteristics like the molecular size and the side chain distribution. To determine the ideal characteristics necessary for personal care products, the Nano3Bio partner company Cosphatec is testing different chitosan types provided by other consortium partners. By combining different chitosan types, Cosphatec is able to produce a cream formulation in which no other thickener is needed. At the same time, the antimicrobial stability is kept while reducing preservation significantly to a minimum of the usual concentration. Within upcoming research activities of Nano3Bio, it is even aimed to further optimise these results. So far, the heterogeneity of currently available chitosan was one of the main hurdles for establishing frequent usage of this raw material. A benefit of the ability to produce chitosan biotechnologically will be the possible control of the process achievable only through exact knowledge of the underlying biological mechanisms. In this way, Nano3Bio aims to manufacture mass tailored chitosan with properties fine tuned to the desired applications in order to satisfy the corresponding market demand with a reliable and reproducible quality.(Nano3Bio consortium partner in charge: Cosphatec)

Learn more about Cosphatec's achievement within Nano3Bio from the short video below:

Low-cost protein engineering technology developed

Synthetic biology researchers from academia and industry alike now have access to low-cost genetic material to help identify protein variants. Developed in part through research funding from the European Union’s Nano3Bio project, the GeneArt Strings DNA Libraries tool makes protein engineering, normally an expensive endeavor, attainable to cost-sensitive customers.

The new GeneArt Strings DNA Libraries is complementary to Thermo Fisher’s existing technology currently used for protein engineering by a process known as directed evolution. This method is designed to identify protein variants with improved properties such as enhanced function, better stability or properties that demonstrate novel enzymatic activity. Proteins engineered with enhanced enzymatic activity have many applications in daily life. As an example, enzymes found in many laundry detergents must be engineered to remain stable and active in hot, detergent-rich water, which are very different conditions compared to their natural environment.

The process of screening and engineering proteins is cost- and labor-intensive. The starting point is a DNA library – a collection of variants of the original DNA sequence encoding the protein of interest. These libraries contain thousands to billions of variants and serve to produce the protein variants that enter the screening procedure.

The expensive screening technology used to identify improved protein variants is compounded by the high cost associated with the meticulous process required to produce high-quality DNA libraries. However, research funded through the Nano3Bio project has enabled implementation of novel technologies and a production workflow to lower the cost of library production. Some of the first researchers to receive these new libraries are Nano3Bio consortium members from IQS in Barcelona, who are using them to develop enzyme variants that can produce novel chitosan oligomer types. The Nano3Bio project is funded by the European Union.

Caption (as to the below figure): Steps to improve proteins using directed evolution technology. Research by the EU funded project Nano3Bio makes this process, launched by Thermo Fisher Scientific, more accessible.

Protocol for production of chitosans with defined structures

In order to be able to explore the benefits of chitosans in depth, there is the need of obtaining it with a defined structure. In order to do so, one of Nano3Bio’s strategies is to combine enzymatic processes with chemical transformations. For this purpose, the project has dealt with the chemical activation of compounds for the in vitro preparation of controlled chitooligo-saccharide polymers (organic compounds formed by the repetitive linkage of small molecules called monomers) by certain (enzyme-catalyzed) reactions. Materials readily available from natural resources are transformed through chemical reactions into monomers designed to self-condense in a well-defined manner when reacting in the presence of an engineered enzyme. In order to obtain the desired monomers, specifically developed conditions are applied onto the starting materials followed by a treatment that allows the isolation of the product. Thus, other minor impurities that could be detrimental for the quality of the polymer or could even prevent the polymerization reaction are removed from the monomers. These reaction conditions developed during the project do not only allow the preparation of simple molecules, but also of structurally more complex compounds that may confer special properties to the polymers prepared from them. Enantia, a Nano3Bio partner company seated in Barcelona, mainly executed these activities.(Nano3Bio consortium partner in charge: Enantia)

Learn more about this Nano3Bio achievement in the following short film:

Learn more about chitosans as important renewable biomaterials

Huge Nano3Bio potentials

While the oil is slowly but surely running out, renewable resources are becoming increasingly important. In future, the biological production of raw materials has to play an even greater role to meet the needs in a more efficient manner, with less resource consumption and environmental pollution. Our international consortium of researchers and companies now rises to this challenge. Learn more ...

Manifold benefits and connecting factors

The potential of functional bio-polymers – Nano3Bio's main object of investigation and engineering – is evident. Nano3Bio provides significant chances as well as natural connections to a wide variety of target groups and application fields. Science, industry, society and presumably also the environment will benefit. Learn more ...

Paper on legal analysis available

High-value news for those who are interested in legal aspects published in a paper as a result of Nano3Bio: "Biotechnologically produced chitosan for nanoscale products. A legal analysis" (2018). Read paper or download PDF ...

Factsheet for a brief overview on Nano3Bio

Get our new up-to-date factsheet to learn basics about the Nano3Bio approach, its achievements and potentials. Download now ...